Despite the prominent role for tissue factor (TF)in various physiological processes, primarily in those related to blood coagulation, there is a lack of information with respect to the structure and function of natural TF forms existing in vivo. Most experimental data related to TF have been derived using various forms of recombinant proteins. Our preliminary data indicate that monocytic TF present on the native membrane is 150-400-fold more active than any TF, recombinant or natural, presented on an artificial membrane. The primary goal of this proposal is to study natural human TF from multiple sources by evaluating their structural and functional properties and by identifying the cell membrane component(s) responsible for high TF activity. Substantial amounts of placental, monocyte and mtcroparticle TF proteins will be purified by immunoaffinity methods. Characterization of purified TF species will utilize immunoassays, structural, biochemical and functional assays as well as structure-function analyses. Functional characterization of TF proteins both purified and presented on native membranes will include factor Vila-driven reactions in one enzyme-one substrate systems (fluorogenic assays, extrinsic factor Xase and factor IX activation) followed by more complex systems, such as TF-initiated thrombin generation in synthetic coagulation proteome and whole blood. The posttranslational modifications of the purified TF species will be characterized using deglycosylation, tryptic digestion, mass-spectroscopy and sequencing. The influence of these modifications on TF activity will be analyzed. Purified human TF from placenta will be used as a standard in all evaluations, functional and structural. Three different species of recombinant human TF will be used for comparison as well, i.e. TF^ea (full-length), TFi_242 (lacking the cytoplasmic domain), and TFi.2is (soluble; extracellular domain only). We will also attempt to understand the mechanism underlying high monocyte TF in situ activity by purifying, analyzing and evaluating the component(s) of the monocyte membrane, lipid rafts and monocyte-derived microparticles. The data accumulated during this study will expand our knowledge related to the structural and functional properties of natural human TF. New knowledge linking TF structure and environment with functional activity will be obtained. Relevance: This project will lead to a better understanding of the mechanisms regulating the activity of tissue factor, a key protein in the initiation of blood clotting and an emerging link between inflammation and thrombosis.